The invention relates to the genetic manipulation of plants, particularly to transforming plants with genes that enhance disease resistance, and breeding program for screening tolerance/resistance lines.
Disease in plants is caused by biotic and abiotic causes. Biotic causes include fungi, viruses, bacteria, and nematodes. Of these, fungi are the most frequent causative agent of disease on plants. Abiotic causes of disease in plants include extremes of temperature, water, oxygen, soil pH, plus nutrient-element deficiencies and imbalances, excess heavy metals, and air pollution.
A host of cellular processes enables plants to defend themselves from disease caused by pathogenic agents. These processes apparently form an integrated set of resistance mechanisms that is activated by initial infection and then limits further spread of the invading pathogenic microorganism.
Subsequent to recognition of a potentially pathogenic microbe, plants can activate an array of biochemical responses. Generally, the plant responds by inducing several local responses in the cells immediately surrounding the infection site. The most common resistance response observed in both non-host and race-specific interactions is termed the xe2x80x9chypersensitive responsexe2x80x9d (HR). In the hypersensitive response, cells contacted by the pathogen, and often neighboring cells, rapidly collapse and dry in a necrotic fleck. Other responses include the deposition of callose, the physical thickening of cell walls by lignification, and the synthesis of various antibiotic small molecules and proteins. Genetic factors in both the host and the pathogen determine the specificity of these local responses, which can be very effective in limiting the spread of infection.
The hypersensitive response in many plant-pathogen interactions results from the expression of a resistance (R) gene in the plant and a corresponding avirulence (avr) gene in the pathogen. (Flor (1971) Ann. Rev. Phytopath. 9:274). This interaction is associated with the rapid, localized cell death of the hypersensitive response. R genes that respond to specific bacterial, fungal, or viral pathogens, have been isolated from a variety of plant species and several appear to encode cytoplasmic proteins.
The resistance gene in the plant and the avirulence gene in the pathogen often conform to a gene-for-gene relationship. That is, resistance to a pathogen is only observed when the pathogen carries a specific avirulence gene and the plant carries a corresponding or complementing resistance gene. The theory has been developed by genetic studies of plant/pathogen interactions in a variety of plant systems (Keen (1990) Ann. Rev. Genet. 24:447-463). Because avrR gene-for-gene relationships are observed in many plant-pathogen systems and are accompanied by a characteristic set of defense responses, a common molecular mechanism underlying avrR gene mediated resistance has been postulated. A simple model which has been proposed is that pathogen avr genes directly or indirectly generate a specific molecular signal (ligand) that is recognized by cognate receptors encoded by plant R genes.
Both plant resistance genes and corresponding pathogen avirulence genes have been cloned. The plant kingdom contains thousands of R genes with specific specificities for viral, bacterial, fungal, or nematode pathogens. Although there are differences in the defense responses induced during different plant-pathogen interactions, some common themes are apparent among R gene-mediated defenses. The function of a given R gene is dependent on the genotype of the pathogen. Plant pathogens produce a diversity of potential signals, and in a fashion analogous to the production of antigens by mammalian pathogens, some of these signals are detectable by some plants.
Recently a number of R genes have been isolated from various plant species that interact with different pathogens (avr) genes (Bent (1996) Plant Cell 8:1757-1771; Hammond-Kosack et al. (1997) Ann. Rev. Plant Physiol. Plant Mol. Biol. 48:575-607; Ellis etal. (1988) Cur. Opin. in Plant Path. 1:288-293). The recent development of new methods for gene isolation in plants has permitted isolation of many R genes from other plant species (Yu et al. (1996) Proc. Natl. Acad. Sci. USA 93:11751-11756; Leister et al. (1998) Proc. Natl. Acad. Sci. USA. 95:370-375). Although the products of most avirulence genes show very little homology with each other, the reported R genes have conserved functional domains such as the protein kinase (PK) domain, leucine-rich regions (LRR), nucleotide-binding sites (NBS) Bent (1996) Plant Cell 8:1757-1771; Ellis et al. (1988) Cur. Opin. in Plant Path. 1:288-293).
The R genes can be classified into four main classes based on the structure of R gene products (Bent (1996) Plant Cell 8:1757-1771; Ellis et al. (1988) Cur. Opin. in Plant Path. 1:288-293; Hammond-Kosack et al. (1997) Ann. Rev. Plant Physiol. Plant Mol. Biol. 48:575-607; NBS-LRR (nucleotide-binding site-Leucine-rich repeat), LRR-TM-PK (Leucine-rich repeat-transmembrane domain-protein kinase), LRR-TM (Leucine-rich repeat-transmembrane domain), and PK (protein kinase). The largest number of characterized R proteins is the NBS-LRR type. These group genes can be recognized into two subgroups by the presence or absence of an amino-terminal region (TIR domain). TIR domain has sequence and structural similarity to the cytoplasmic signaling domains of Toll and interleukin-1 receptor (Baker et al. (1997) Science 276:726-733; Parker et al. (1997) Plant Cell 9:879-894).
The first subgroup includes tobacco TMV N gene, Hammond-Kosack et al. (1997) Ann. Rev. Plant Physiol. Plant Mol. Biol. 48:575-607, L6 (flax rust resistance), Hammond-Kosack et al. (1997) Ann. Rev. Plant Physiol. Plant Mol. Biol. 48:575-607, and RPP5 (downy mildew resistance), Parker et al. (1997) Plant Cell 9:879-894. The second subgroup without TIR domain includes RPS2, RPM1 Hammond-Kosack et al. (1997) Ann. Rev. Plant Physiol. Plant Mol. Biol. 48:575-607, and Prf Salmeron et al. (1996) Cell 86:123-133. These three R genes contain an amino-terminal leucine zipper (LZ) domain. The LZ domain may involve in protein-protein interaction in the signaling pathways. The proteins in these two subclasses signal through different pathways. Proteins in the TIR class signal via a pathway that includes EDS1 (Parker et al. (1996) Plant Cell 8:2033-2046) whereas the other class signals through a pathway that includes NDR1 gene (Centry et al. (1997) Science 278:1963-1965). The function of NBS may relate to HR and evidence has shown that NBS of RPS2 binds nucleotide (Ellis et al. (1988) Curr. Opin. in Plant Path. 1:288-293). Many NBS-LRR plant genes have been identified through plant genome sequencing projects (Botella et al. (1997) Plant J. 12:1197-1121), and by PCR using degenerate primers based on the NBS motifs. Yu et al. (1996) Proc. Natl. Acad Sci. USA 93:11751-11756; Leister et al. (1998) Proc. Natl. Acad. Sci. USA. 95:370-375.
Both LRR-TM-PK and PK classes of R genes contain kinase domain. Ligand binding activates the protein kinase domain. The first receptor-like protein kinase (RLK) gene is maize ZmPK1 that was isolated using degenerate primers to the kinase domain (Walker et al. (1990) Nature 345:743-746). Since then, a number of RLKs have been isolated from plants. The plant RLKs belongs to serine/threonine kinase family except ZmPK1. All of the plant RLKs can be classified according to the features of the predicted extracellular domain and transmambrane domain, such as LRR and TM. The function of kinase in defense signaling pathways has been dissected in the Pto/AvrPto interactions (Oldroyd et al. (1988) Proc. Natl. Acad. Sci. USA 95:10300-10305; Martin et al. (1996) Molecular Aspects of Pathogenicity and Resistance: Requirement for Signal Transduction, (pages 163-186). In the signal transduction, Pto, together with Prf, is required for the hypersensitive response induction after AvrPto perception. Pto also interacts with a family of transcriptional factors-Pti4, Pti5, and Pti6- that binds to a conserved cis-element present in promoter region of many genes encoding PR proteins (Zhou et al. (1997) EMBO J. 16:3207-3218). Expression of PR proteins is specifically enhanced upon Pto-Avrpto recognition in transgenic tobacco proteins (Zhou et al. (1997) EMBO J. 16:3207-3218).
In general, an R gene provides resistance against only some strains of a particular pathogen species. However, it has been demonstrated that R genes can be functional in heterologous systems (Thilmony et al. (1995) Plant Cell 7:1529-1536; Parker et al. (1996) Plant Cell 8:2033-2046). Furthermore, overexpression of R gene Prf activates pathways in a pathogen-independent manner and leads to the activation of systemic acquired resistance (SAR). The transgene-induced SAR has implications for the generation of broad-spectrum disease resistance in agricultural crop plants (Oldroyd et al. (1988) Proc. Natl. Acad. Sci. USA 95:10300-10305). One example of a strain-nonspecific resistance gene, mlo, has been cloned (Buschges et al. (1997) Cell 88:695-705).
As noted, among the causative agents of infectious disease of crop plants, the phytopathogenic fungi play the dominant role. Phytopathogenic fungi cause devastating epidemics, as well as causing significant annual crop yield losses. All of the approximately 300,000 species of flowering plants are attacked by pathogenic fungi. However, a single plant species can be host to only a few fungal species, and similarly, most fungi usually have a limited host range.
Plant disease outbreaks have resulted in catastrophic crop failures that have triggered famines and caused major social change. Generally, the best strategy for plant disease control is to use resistant cultivars selected or developed by plant breeders for this purpose. However, the potential for serious crop disease epidemics persists today, as evidenced by outbreaks of the Victoria blight of oats and southern corn leaf blight. Accordingly, molecular methods are needed to supplement traditional breeding methods to protect plants from pathogen attack.
Compositions and methods for creating or enhancing resistance to plant pests in a plant are provided. Compositions of the invention comprise novel disease resistance analogs from sunflower and variants of such resistance genes. The methods involve stably transforming a plant with nucleotide sequences of the invention operably linked with a promoter capable of driving expression of a gene in a plant cell. The sunflower R gene analogs and variants thereof may be used to modulate expression of the resistance gene analogs in plants and to engineer plants with broad-spectrum disease resistance. The methods of the invention find use in controlling plant pests, including fungal pathogens, viruses, nematodes, insects, and the like.
The compositions of the invention are additionally useful as genetic markers. Such genes can be used to facilitate the sunflower breeding programs for disease resistance.
Transformed plants and seeds, as well as methods for making such plants and seeds are additionally provided.